(1) Field of the Invention
The invention relates to a method and device for use with a spectrophotometer, for fluorimetric determination of a biological, chemical or physical parameter of a sample, utilizing at least two different luminescent materials, the first of which is sensitive to the parameter at least with respect to luminescence intensity, and the second of which is insensitive to the parameter at least with respect to luminescence intensity and decay time.
The invention relates in particular to a new principle of optical detection of chemical parameters with the use of optical sensors, based on phase shift- and time-resolved measurements. The modulation frequencies used are between 0.5 and 5 MHz and may be detected with the use of low-cost optical semiconductor components.
(2) Description of the Related Art
As is known from the literature and practical experience with optical sensors, determining decay time instead of intensity as a measurement variable has certain practical advantages in luminescence measurements. Fluctuations in the optical system, for instance, will hardly or not at all interfere with decay time. Neither changes in intensity of the light source and sensitivity of the photodetector nor signal losses due to bent fibers or influences on signal intensity by changes in the sensor geometry will have any effects on the measured signal. This will also apply to undefined optical properties of the sample (such as turbidity, intrinsic colour, or refractive index), which could cause problems in intensity measurements.
Further, in many cases the measured signal is largely independent of the concentration of the indicator in the sensitive layer. For this reason a slight degree of photo-decomposition or leaching is less critical.
In the literature a large number of measuring principles have been proposed for monitoring chemical parameters by means of decay time. One of the most frequently used methods is dynamic luminescence quenching, i.e., non-radiative deactivation of the excited state of a luminescence indicator by the analyte. This approach is used for optical detection of molecular oxygen and the detection of halide and heavy metal ions (1).
A further method of deactivation utilizes photo-induced electron transfer in a single indicator molecule. In this instance (shortly called PET) the luminescence indicator is included in different forms, only one of which (i.e., acid form or with bonded metal ion) features strong luminescence and long lifetime. In the other form (i.e., basic form or without bonded metal ion) the indicator has a free electron pair, which can deactivate the excited state without radiation. As a consequence, both decay time and luminescence quantum efficiency will decrease. This principle may be employed with optical pH measurement, or optical ionic sensor technology (2).
Another proposed method of decay time measurement utilizes the effect that certain pH indicators exhibit different luminescence intensities and different, though defined, decay times for their protonated and deprotonated states. Such indicators include derivatives of seminaphthofluorescein, for example. In such instance luminescence of the acid and basic form is monitored simultaneously. The respective (pH-dependent) ratio of the two intensities will yield a mean decay time which can be measured (3). A precondition for this method is that both forms of an indicator are luminescent, and that their absorption and emission spectra show significant overlap.
It should be noted that in most of the measuring principles cited above, measured decay times are in the range of a few nanoseconds as a rule. Accurate measurements of decay times in the lower nanosecond region necessitate expensive instrumentation, however, as they require not only very fast circuitry and high modulation frequencies but also fast light sources and detectors. It seems unlikely for this reason that low-cost instruments utilizing optical semiconductor components, such as light emission diodes and photodiodes, will be developed for this type of sensors in the near future. If optical sensors are to be used for a wide range of applications, however, inexpensive instrumentation is indispensable. For this reason there is considerable interest in decay time sensors whose measuring range is in the microsecond or even millisecond region. So far such sensors have been developed and put to practical use almost exclusively for optical oxygen measurement, where indicators with decay times of up to several milliseconds are employed.
A recent approach to develop new, long-life decay time sensors makes use of radiationless energy transfer from a luminescent donor molecule, whose photophysical properties are not affected by the analyte, to a dye indicator referred to as acceptor, which is sensitive to the analyte. Depending on the respective analyte concentration the absorption spectrum of the acceptor must overlap to a varying extent with the emission spectrum of the donor. Suitable luminescent donors are transition metal complexes with ruthenium(II), ruthenium(I), or osmium and iridium as central atom. These compounds feature long lifetimes (some 100 nsec to a few microseconds) and high quantum yields. This novel approach was first proposed by Lakowicz and has recently been employed with optical pH sensors. In principle, such optodes could be put to use for pCO2, NH3, and ion detection in a similar manner (4,5).
One serious problem with the practical use of such sensors arises from the fact that the rate of energy transfer, and hence measurable mean decay time will significantly depend not only on the distance and positioning of donor and acceptor molecule, but also on the concentration of the acceptor in the matrix. As a consequence, each change in distribution and distance of the indicators in the matrix will lead to changes in the sensor characteristic. Swelling of the matrix, in particular, will constitute a grave problem.
Another problem is caused by the influence of oxygen on the sensors. Since the luminescence of the long-life donors used often is substantially quenched by oxygen, oxygen concentration must be included in the measurement and the measured signal must be corrected. In addition, reactive singlet oxygen is produced during this process in the membrane, which will accelerate photo-decomposition of the immobilized indicators, thus reducing both storage and long-term stability. As a consequence, one of the classical advantages of decay time measurement is lost.
A device of the above described type is disclosed in U.S. Pat. No. 5,102,625, where the intensities of two luminescent materials are separately measured by means of two separate measuring channels. The intensity ratio of the two luminescent materials is used as the final signal for monitoring of the parameter. The luminescence decay times do not enter the measurement. The two luminescent materials have differing spectral regions.
It is an object of the present invention to propose a method and device for fluorimetric determination of the parameter of a sample, which combine high measuring accuracy and comparative simplicity of instrumentation.
The invention describes a new measuring principle permitting fluorimetric determination of various chemical, physical, and biological parameters with the use of time-resolved methods and phase modulation techniques. The invention permits effective referencing of the intensity signals of the majority of fluorescence.sensors described in the literature by admixing a long-life luminescent material. For this purpose two different luminescent materials are jointly co-immobilized in the sensor. The sum signal is derived from a luminescence signal with constant long-life decay time (at least some hundred nanoseconds) and a short-lived fluorescence signal. Whereas the parameters of long-lived luminescence are not affected by the analyte, the intensity of the co-immobilized, short-life luminescent material will vary with the respective analyte concentration. As the phase shift xcfx86m obtained by phase modulation techniques depends exclusively on the ratio of the partial intensities of the two individual luminescent materials, this parameter will directly reflect the intensity of the luminescent material sensitive to the parameter. The invention thus is concerned with a new method of internally referencing the signal intensity of fluorophores without the necessity of a second light source or a second photodetector. Provided that the distribution of the two luminescent materials is maintained constant during manufacture, xcfx86m will depend exclusively on the physical or chemical parameter being monitored, whilst variations in the optoelectronic system, in losses in the fiber optics and in the optical properties of the sample will have no influence on the signal.
Preferably, both luminescent materials will absorb light in the same range of wavelengths, which will enable them to be excited into luminescence by means of a single light source. Emission spectra will preferably be in the same spectral region. It will be possible, for example, to excite both luminescent materials with blue light at a wavelength of 450 nm, one luminescent material emitting green light at 520 nm and the other one red light at 600 nm, as both signals can still be measured with one and the same detector. It will also be possible, however, to simultaneously measure the luminescence of two luminescent materials whose emission spectra differ from each other significantly.
The measuring method described has the advantage that the long-life luminescent material need not exhibit analyte-specific response but solely acts as carrier of a constant background signal with long decay time, which is modulated by the short-life luminescent material. For this reason a large number of phosphorescent compounds described in the literature are suitable for this purpose.
The long-life luminescent material need not interact with sample, analyte, or fluorescence indicator, and can thus be immobilized such that it is inert to all sample components, which will exclude a priori any potential interference by chemical parameters.